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1.
Rev Med Suisse ; 17(732): 624-629, 2021 Mar 31.
Artículo en Francés | MEDLINE | ID: mdl-33793099

RESUMEN

The development of in vivo skin imaging technologies has been booming for several decades. Their advantages are indisputable, especially as they are non-invasive. Their place is already well established in onco-dermatology and it is just a question of time for them to be used with success in other fields of dermatology, including pediatric dermatology. In this paper we will discuss 3 of these skin imaging techniques used in dermatology at the CHUV, including Optical Coherence Tomography (OCT), Reflectance Confocal Microscopy (RCM) and the most recent: Line-field Confocal Optical Coherence Tomography (LC-OCT).


Asunto(s)
Dermatología , Neoplasias Cutáneas , Niño , Humanos , Microscopía Confocal , Piel/diagnóstico por imagen , Neoplasias Cutáneas/diagnóstico por imagen , Tomografía de Coherencia Óptica
2.
Adv Exp Med Biol ; 1310: 1-30, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834430

RESUMEN

Confocal laser scanning microscopy (CLSM) and related microscopic techniques allow a unique and versatile approach to image and analyze living cells due to their specificity and high sensitivity. Among confocal related techniques, fluorescence correlation methods, such as fluorescence correlation spectroscopy (FCS) and dual-color fluorescence cross-correlation spectroscopy (FCCS), are highly sensitive biophysical methods for analyzing the complex dynamic events of molecular diffusion and interaction change in live cells as well as in solution by exploiting the characteristics of fluorescence signals. Analytical and quantitative information from FCS and FCCS coupled with fluorescence images obtained from CLSM can now be applied in convergence science such as drug delivery and nanomedicine, as well as in basic cell biology. In this chapter, a brief introduction into the physical parameters that can be obtained from FCS and FCCS is first provided. Secondly, experimental examples of the methods for evaluating the parameters is presented. Finally, two potential FCS and FCCS applications for convergence science are introduced in more detail.


Asunto(s)
Microscopía Confocal , Color , Difusión , Espectrometría de Fluorescencia , Coloración y Etiquetado
3.
Adv Exp Med Biol ; 1310: 31-58, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834431

RESUMEN

Number and brightness (N&B) analysis helps to visualize protein oligomer and its localization in a living cell. N&B analysis provides apparent brightness, which reflects the oligomeric state of a fluorescently labeled protein, by analyzing the temporal intensity fluctuation at each pixel. N&B analysis is useful in understanding the dynamic oligomerization in signal transduction and neurodegenerative diseases. Furthermore, it also helps in gaining useful insights regarding the controlling mechanisms in protein function. In this chapter, we describe the basic theory and notations of N&B analysis implemented with confocal laser scanning microscopy for quantitative analyses.


Asunto(s)
Transducción de Señal , Proteínas Fluorescentes Verdes , Microscopía Confocal , Multimerización de Proteína , Espectrometría de Fluorescencia
4.
Adv Exp Med Biol ; 1310: 153-186, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33834437

RESUMEN

Intravital microscopy has emerged as a powerful technique for the fluorescent visualization of cellular- and subcellular-level biological processes in vivo. However, the size of objective lenses used in standard microscopes currently makes it difficult to access internal organs with minimal invasiveness in small animal models, such as mice. Here we describe front- and side-view designs for small-diameter endoscopes based on gradient-index lenses, their construction, their integration into laser scanning confocal microscopy platforms, and their applications for in vivo imaging of fluorescent cells and microvasculature in various organs, including the kidney, bladder, heart, brain, and gastrointestinal tracts, with a focus on the new techniques developed for each imaging application. The combination of novel fluorescence techniques with these powerful imaging methods promises to continue providing novel insights into a variety of diseases.


Asunto(s)
Endoscopía , Lentes , Animales , Microscopía Intravital , Riñón , Ratones , Microscopía Confocal
5.
Nat Commun ; 12(1): 1778, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741954

RESUMEN

Memory reconsolidation, the process by which memories are again stabilized after being reactivated, has strengthened the idea that memory stabilization is a highly plastic process. To date, the molecular and cellular bases of reconsolidation have been extensively investigated particularly within the hippocampus. However, the role of adult neurogenesis in memory reconsolidation is unclear. Here, we combined functional imaging, retroviral and chemogenetic approaches in rats to tag and manipulate different populations of rat adult-born neurons. We find that both mature and immature adult-born neurons are activated by remote memory retrieval. However, only specific silencing of the adult-born neurons immature during learning impairs remote memory retrieval-induced reconsolidation. Hence, our findings show that adult-born neurons immature during learning are required for the maintenance and update of remote memory reconsolidation.


Asunto(s)
Aprendizaje/fisiología , Consolidación de la Memoria/fisiología , Memoria a Largo Plazo/fisiología , Neuronas/fisiología , Animales , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Hipocampo/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Microscopía Confocal , Neuronas/metabolismo , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Ratas Sprague-Dawley , Factores de Tiempo
6.
Nat Commun ; 12(1): 1773, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741995

RESUMEN

The exploration of artificial luminogens with bright emission has been fully developed with the advancement of synthetic chemistry. However, many of them face problems like weakened emission in the aggregated state as well as poor renewability and sustainability. Therefore, the development of renewable and sustainable luminogens with anti-quenching function in the solid state, as well as to unveil the key factors that influence their luminescence behavior become highly significant. Herein, a new class of natural rosin-derived luminogens with aggregation-induced emission property (AIEgens) have been facilely obtained with good biocompatibility and targeted organelle imaging capability as well as photochromic behavior in the solid state. Mechanistic study indicates that the introduction of the alicyclic moiety helps suppress the excited-state molecular motion to enhance the solid-state emission. The current work fundamentally elucidates the role of alicyclic moiety in luminogen design and practically demonstrates a new source to large-scalely obtain biocompatible AIEgens.


Asunto(s)
Materiales Biocompatibles/química , Colorantes Fluorescentes/química , Luminiscencia , Resinas de Plantas/química , Animales , Materiales Biocompatibles/farmacología , Células COS , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Escherichia coli/efectos de los fármacos , Colorantes Fluorescentes/farmacología , Microscopía Confocal , Estructura Molecular , Movimiento (Física) , Imagen Óptica/métodos , Orgánulos/química , Orgánulos/metabolismo , Resinas de Plantas/farmacología , Relación Estructura-Actividad
7.
Nat Commun ; 12(1): 1814, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753734

RESUMEN

The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson's disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-ß structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.


Asunto(s)
Amiloide/metabolismo , Cuerpos de Inclusión/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/química , Animales , Calcio/metabolismo , Línea Celular Tumoral , Células Cultivadas , Humanos , Cinética , Microscopía Confocal , Enfermedad de Parkinson/metabolismo , Agregación Patológica de Proteínas , Multimerización de Proteína , Ratas Sprague-Dawley , alfa-Sinucleína/química
8.
Nat Commun ; 12(1): 1807, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753743

RESUMEN

Mitochondria-lysosome contacts are recently identified sites for mediating crosstalk between both organelles, but their role in normal and diseased human neurons remains unknown. In this study, we demonstrate that mitochondria-lysosome contacts can dynamically form in the soma, axons, and dendrites of human neurons, allowing for their bidirectional crosstalk. Parkinson's disease patient derived neurons harboring mutant GBA1 exhibited prolonged mitochondria-lysosome contacts due to defective modulation of the untethering protein TBC1D15, which mediates Rab7 GTP hydrolysis for contact untethering. This dysregulation was due to decreased GBA1 (ß-glucocerebrosidase (GCase)) lysosomal enzyme activity in patient derived neurons, and could be rescued by increasing enzyme activity with a GCase modulator. These defects resulted in disrupted mitochondrial distribution and function, and could be further rescued by TBC1D15 in Parkinson's patient derived GBA1-linked neurons. Together, our work demonstrates a potential role of mitochondria-lysosome contacts as an upstream regulator of mitochondrial function and dynamics in midbrain dopaminergic neurons in GBA1-linked Parkinson's disease.


Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Glucosilceramidasa/genética , Lisosomas/genética , Mitocondrias/genética , Mutación , Enfermedad de Parkinson/genética , Células Cultivadas , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/ultraestructura , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Glucosilceramidasa/metabolismo , Humanos , Hidrólisis , Lisosomas/metabolismo , Lisosomas/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Enfermedad de Parkinson/metabolismo , Imagen de Lapso de Tiempo/métodos , Proteínas de Unión al GTP rab/metabolismo
9.
Nat Commun ; 12(1): 1932, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771998

RESUMEN

The physical distance between presynaptic Ca2+ channels and the Ca2+ sensors triggering the release of neurotransmitter-containing vesicles regulates short-term plasticity (STP). While STP is highly diversified across synapse types, the computational and behavioral relevance of this diversity remains unclear. In the Drosophila brain, at nanoscale level, we can distinguish distinct coupling distances between Ca2+ channels and the (m)unc13 family priming factors, Unc13A and Unc13B. Importantly, coupling distance defines release components with distinct STP characteristics. Here, we show that while Unc13A and Unc13B both contribute to synaptic signalling, they play distinct roles in neural decoding of olfactory information at excitatory projection neuron (ePN) output synapses. Unc13A clusters closer to Ca2+ channels than Unc13B, specifically promoting fast phasic signal transfer. Reduction of Unc13A in ePNs attenuates responses to both aversive and appetitive stimuli, while reduction of Unc13B provokes a general shift towards appetitive values. Collectively, we provide direct genetic evidence that release components of distinct nanoscopic coupling distances differentially control STP to play distinct roles in neural decoding of sensory information.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Modificados Genéticamente , Conducta Apetitiva/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Interneuronas/metabolismo , Interneuronas/fisiología , Proteínas de la Membrana/genética , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Sinapsis/metabolismo , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismo
10.
Nat Commun ; 12(1): 1924, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772006

RESUMEN

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Demencia Frontotemporal/genética , Proteínas Mitocondriales/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Demencia Frontotemporal/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Homología de Secuencia de Aminoácido
11.
Nat Commun ; 12(1): 1901, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772008

RESUMEN

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.


Asunto(s)
Arabidopsis/metabolismo , Microscopía Confocal/métodos , Vacuolas/metabolismo , Red trans-Golgi/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clatrina/genética , Clatrina/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Electrónica de Transmisión , Plantas Modificadas Genéticamente , Transporte de Proteínas , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Vacuolas/ultraestructura , Red trans-Golgi/ultraestructura
12.
Nat Commun ; 12(1): 1913, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33772014

RESUMEN

Diffusion is a major molecular transport mechanism in biological systems. Quantifying direction-dependent (i.e., anisotropic) diffusion is vitally important to depicting how the three-dimensional (3D) tissue structure and composition affect the biochemical environment, and thus define tissue functions. However, a tool for noninvasively measuring the 3D anisotropic extracellular diffusion of biorelevant molecules is not yet available. Here, we present light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP), which noninvasively determines 3D diffusion tensors of various biomolecules with diffusivities up to 51 µm2 s-1, reaching the physiological diffusivity range in most biological systems. Using cornea as an example, LiFT-FRAP reveals fundamental limitations of current invasive two-dimensional diffusion measurements, which have drawn controversial conclusions on extracellular diffusion in healthy and clinically treated tissues. Moreover, LiFT-FRAP demonstrates that tissue structural or compositional changes caused by diseases or scaffold fabrication yield direction-dependent diffusion changes. These results demonstrate LiFT-FRAP as a powerful platform technology for studying disease mechanisms, advancing clinical outcomes, and improving tissue engineering.


Asunto(s)
Córnea/metabolismo , Espacio Extracelular/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Tendones/metabolismo , Animales , Anisotropía , Colágeno/química , Colágeno/metabolismo , Difusión , Análisis de Fourier , Microscopía Confocal/métodos , Microscopía Electrónica de Rastreo/métodos , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Porcinos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química
13.
J Vis Exp ; (169)2021 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-33749674

RESUMEN

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining and imaging of thick brain samples. However, this advanced technology has not yet been used for whole-mount retinas. Although the retina is partially transparent, its thickness of approximately 200 µm (in mice) still limits the penetration of antibodies into the deep tissue as well as reducing light penetration for high-resolution imaging. Here, we adapted the CLARITY method for whole-mount mouse retinas by polymerizing them with an acrylamide monomer to form a nanoporous hydrogel and then clearing them in sodium dodecyl sulfate to minimize protein loss and avoid tissue damage. CLARITY-processed retinas were immunostained with antibodies for retinal neurons, glial cells, and synaptic proteins, mounted in a refractive index matching solution, and imaged. Our data demonstrate that CLARITY can improve the quality of standard immunohistochemical staining and imaging for retinal neurons and glial cells in whole-mount preparation. For instance, 3D resolution of fine axon-like and dendritic structures of dopaminergic amacrine cells were much improved by CLARITY. Compared to non-processed whole-mount retinas, CLARITY can reveal immunostaining for synaptic proteins such as postsynaptic density protein 95. Our results show that CLARITY renders the retina more optically transparent after the removal of lipids and preserves fine structures of retinal neurons and their proteins, which can be routinely used for obtaining high-resolution imaging of retinal neurons and their subcellular structures in whole-mount preparation.


Asunto(s)
Retina/metabolismo , Coloración y Etiquetado/métodos , Células Amacrinas/fisiología , Animales , Dendritas/fisiología , Neuronas Dopaminérgicas/fisiología , Procesamiento de Imagen Asistido por Computador , Ratones , Microscopía Confocal/métodos , Proteínas del Tejido Nervioso/metabolismo , Receptores AMPA/metabolismo , Refractometría
14.
Methods Mol Biol ; 2265: 47-63, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704704

RESUMEN

In order to protrude within a dense tissue, tumor cells have to develop the ability to digest the extracellular matrix (ECM). Melanoma cells, similarly to other types of tumor cells, form invadopodia, membranous invaginations rich in filamentous actin and several other proteins including matrix metalloproteinases (MMPs). MMPs degrade ECM structural proteins such as collagens, fibronectin, or laminin. Here we describe an assay that allows the detection of gelatinase activity exhibited by tumor cells under 2D conditions and methods to present obtained data in both a quantitative and a qualitative manner.


Asunto(s)
Matriz Extracelular/enzimología , Gelatina/metabolismo , Melanoma/enzimología , Microscopía Confocal/métodos , Actinas/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fluorescencia , Gelatinasas/metabolismo , Humanos , Metaloproteinasas de la Matriz/metabolismo , Melanoma/patología , Imagen Óptica , Podosomas/enzimología , Podosomas/metabolismo , Podosomas/patología
15.
Methods Mol Biol ; 2265: 141-154, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704712

RESUMEN

Three-dimensional (3D) cell culture has allowed a deeper understanding of complex pathological and physiological processes, overcoming some of the limitations of 2D cell culture on plastic and avoiding the costs and ethical issues related to experiments involving animals. Here we describe a protocol to embed single melanoma cells alone or together with primary human lymphatic endothelial cells in a 3D cross-linked matrix, to investigate the invasion and molecular crosstalk between these two cell types, respectively. After fixation and staining with antibodies and fluorescent conjugates, phenotypic changes in both cell types can be specifically analyzed by confocal microscopy.


Asunto(s)
Técnicas de Cocultivo/métodos , Células Endoteliales/metabolismo , Melanoma/metabolismo , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Células Endoteliales/citología , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Melanoma/patología , Microscopía Confocal , Invasividad Neoplásica
16.
Nat Commun ; 12(1): 1708, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33731714

RESUMEN

Gluten, which makes up 85% of endosperm wheat protein, is considered a crucial quality determinant of wheat-based food products. During wheat dough manufacture, the molecular packing of gluten causes formation of large structures that exceed the millimetre scale. However, due to lack of imaging techniques for complex systems composed of giant macromolecules, the entire gluten structure remains unknown. Here, we develop an optical clearing reagent (termed SoROCS) that makes wheat-based products transparent. Combined with two-photon microscopy, we image the three-dimensional (3D) structure of gluten at the size in the millimetre scale and at submicron resolution. Further, we demonstrate how the 3D structure of gluten dramatically changes from a honeycomb-shaped network to sparse large clumps in wheat noodles, depending on the salt added during dough making, thereby reducing stress when compressing the noodle. Moreover, we show that SoROCS can be used for noodle imaging using confocal laser scanning microscopy.


Asunto(s)
Glútenes/química , Imagenología Tridimensional/métodos , Triticum/química , Microscopía Confocal , Microscopía de Fluorescencia por Excitación Multifotónica , Salicilato de Sodio/química , Solventes/química , Almidón/química
17.
J Appl Oral Sci ; 29: e20200733, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33656065

RESUMEN

OBJECTIVES: Enterococcus faecalis (E. faecalis), one of the main pathogens responsible for refractory periapical periodontitis and nosocomial infections, exhibits markedly higher pathogenicity in biofilms. Studies have shown that caseinolytic protease P (ClpP) is involved in biofilm formation. However, to date, few studies have investigated the role of ClpP in the survival of E. faecalis, and in enhancing biofilm formation. Therefore, we investigated the role of ClpP in the formation of E. faecalis biofilms. METHODOLOGY: In our study, we used homologous recombination to construct clpP deleted and clpP complement strains of E. faecalis ATCC 29212. A viable colony counting method was used to analyze the growth patterns of E. faecalis. Crystal violet staining (CV) and confocal scanning laser microscopy (CLSM) were used to characterize biofilm mass formation and scanning electron microscopy (SEM) was used to observe the biofilm microstructure. Data was statistically analyzed via Student's t-test or one-way analysis of variance (ANOVA). RESULTS: The results exhibited altered growth patterns for the clpP deletion strains and depleted polysaccharide matrix, resulting in reduced biofilm formation capacity compared to the standard strains. Moreover, ClpP was observed to increase biofilm formation in E. faecalis. CONCLUSION: Our study shows that ClpP can increase biofilm formation in E. faecalis and emphasizes the importance of ClpP as a potential target against E. faecalis.


Asunto(s)
Biopelículas , Enterococcus faecalis , Endopeptidasa Clp , Humanos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Péptido Hidrolasas , Virulencia
18.
J Vis Exp ; (168)2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-33645587

RESUMEN

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.


Asunto(s)
Coloración y Etiquetado , Papilas Gustativas/citología , Animales , Recuento de Células , Disección , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratones , Microscopía Confocal , Neuronas/citología , Paladar (Hueso)/citología , Paladar (Hueso)/inervación
19.
Methods Mol Biol ; 2218: 209-218, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606234

RESUMEN

The combination of immunohistochemistry and confocal laser microscopy enables the observation of cellular structures and protein localization within cells using whole-mount tissues. However, such high-resolution imaging requires several steps, such as proper dissection before fixation and antibody staining, and the appropriate positioning of tissues on a glass slide for observation. Here, we describe the method developed by our laboratory for the immunohistochemistry of medaka embryonic and larval gonads, focusing on the dissection and mounting of tissues for confocal laser microscopy. Positioning the gonad just beneath the coverslips is essential to obtain high-resolution images at a level where cellular components of germ cells, such as germ plasm and nuclear structures, can be clearly observed using an oil immersion objective lens.


Asunto(s)
Gónadas/metabolismo , Inmunohistoquímica/métodos , Larva/metabolismo , Microscopía Confocal/métodos , Oryzias/metabolismo , Animales , Células Germinativas/metabolismo , Coloración y Etiquetado/métodos
20.
Methods Mol Biol ; 2235: 1-12, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33576966

RESUMEN

In addition to intravascular dissemination, angiotropic melanoma cells have the propensity to spread along the external surface of blood vessels in a pericytic location, or pericytic mimicry. Such continuous migration without intravasation has been termed "extravascular migratory metastasis" or EVMM. In order to visualize this mechanism of tumor propagation, we used a murine brain melanoma model utilizing green fluorescent human melanoma cells and red fluorescent lectin-tagged murine vessels. This model allows the direct microscopic visualization and mapping of the interaction of melanoma cells with the brain vasculature. In this chapter, we describe the methodology of lectin perfusion to label the entire angioarchitecture in conjunction with confocal microscopy imaging to study the pericyte mimicry of the angiotropic GFP+ melanoma cells.


Asunto(s)
Melanoma/diagnóstico por imagen , Invasividad Neoplásica/diagnóstico por imagen , Imagen Óptica/métodos , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Femenino , Proteínas Fluorescentes Verdes/química , Inmunohistoquímica/métodos , Lectinas/química , Masculino , Melanoma/patología , Ratones , Ratones Desnudos , Microscopía Confocal/métodos , Neovascularización Patológica/patología , Perfusión/métodos , Pericitos , Neoplasias Cutáneas/patología
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